Toxoplasma gondii is an obligate intracellular parasite known to chronically infect a third of the human population. T. gondii is an opportunistic infectious agent that can cause encephalitis and other severe manifestations in AIDS patients. Events required for T. gondii invasion, egress and motility are regulated by calcium signaling processes that include proteins and factors significantly divergent from those of the human host. To exploit these events as potential drug targets would require a better understanding of T. gondii's calcium signaling cascades. A particular family of calcium dependent protein kinases (CDPKs) has garnered special attention, as it is absent from human cells and it is involved in key processes. Through a forward genetic approach we discovered that mutations in TgCDPK3 lead to a delay in calcium ionophore-induced egress (iiEgress), and importantly a defect in the formation of latent stages in the brains of mice. Utilizing a novel screen for protein-protein interactions, we identified several proteins that might act as functional partners or substrates of TgCDPK3, including a protein phosphatase 2C, which we have named TgPP2C4 and another calcium dependent protein kinase TgCDPK2a. It is our hypothesis that TgCDPK3, TgCDPK2a, and TgPP2C4, form part of a co-regulatory network essential for parasite virulence. Our first goal will be to analyze the localization and function of both TgPP2C4 and TgCDPK2a. In addition, we will determine the substrates and functional partners of these two enzymes, which will allow us to define the events and proteins co-regulated by TgCDPK3, TgCDPK2a and TgPP2C4. Our second goal will be to determine the role of TgCDPK3 in virulence. This will be accomplished by analyzing the in vivo dissemination and encystation of parasites lacking TgCDPK3 or its co-regulatory partners TgCDPK2 and TgPP2C. Furthermore, we will identify the substrates of TgCDPK3 during the process of encystation in tissue culture, which will provide mechanistic insight into the function of TgCDPK3 during in vivo propagation and development. In conjunction, our work will shed light on the function of calcium signaling during the life cycle of T. gondii and has the potential to reveal novel targets to combat both acute and chronic toxoplasmosis.